Ultrastructural imaging reveals vascular remodeling in migraine patients.

Migraine is a neurological disorder and one of the most common pain conditions worldwide. Despite its prevalence, the basic biology and underlying mechanisms contributing to the development of migraine are still poorly understood. It is still unclear, for instance, whether the vasculature, both extra and intracranial, plays a significant role in the generation of migraine pain. Neuroimaging data, indeed, have reported conflicting results on blood vessels abnormalities like vasodilation, while functional studies suggest that vessels dysfunction may extend beyond vasodilation. Here we combined light and electron microscopy imaging to investigate the fine structure of superficial temporal (STA) and occipital arteries (OA) from patients that underwent minimally invasive surgery for migraine. Using optical microscopy, we observed that both STA and OA vessels showed marked endothelial thickening and internal elastic lamina fragmentation. In the muscular layer, we found profound shape changes of vascular smooth muscle cells (VSMCs), abundant extracellular matrix, and the presence of clear extracellular vacuoles. The electron microscopy analysis confirmed putative VSMCs infiltrated within the intima layer and revealed a consistent shifting of VSMCs from contractile to a synthetically active phenotype. We also report the presence of (i) abundant extracellular vacuoles filled with fine granular material and membranes, (ii) multilamellar structures, (iii) endosome-like organelles, and (iv) bona fide extracellular vesicles in the matrix space surrounding synthetically active cells. As both the endothelial layer and VSMCs coordinate a variety of vascular functions, these results suggest that a significant vascular remodeling is occurring in STA and OA of migraine patients. Thus, this phenomenon may represent an important target for future investigation designed toward the development of new therapeutic approaches.

Online lessons of human anatomy: Experiences during the COVID-19 pandemic.

The social distancing measures necessitated by the COVID-19 pandemic have resulted in the migration of human anatomy lessons to virtual platforms. Even student communities have had to relocate online. The virtual replacement of visual-spatial and social elements, essential for studying anatomy, has posed particular challenges for educators. Our department used Microsoft Teams, an online communication platform, in conjunction with Visible Body, a 3D anatomical modeling program, EdiErmes online resources, and Leica Acquire for teaching microscopic anatomy. We delivered about 160 h of both synchronous and asynchronous lessons for students on the medical degree program per academic year. In this study, we compare face-to-face and distance teaching in order to define these different approaches better and to evaluate the final student scores. The aim is to debate the relevance of distance learning pedagogy to the design of new online anatomy teaching courses and the development of online learning. Analysis of the final scores showed that anatomy examinations after the online course had a statistically significantly higher average value than those obtained at the end of the face-to-face course. The experience at the University of Genoa shows that distance learning in the teaching of human anatomy was perceived by most students as useful and positive. Distance learning can be an effective support for anatomy teaching, facilitating a different mode of learning in which lessons and study are more sensitive to the individual's schedule and needs. Of course, we should not and cannot exclude face-to-face teaching.

Teaching histology and anatomy online during the COVID-19 pandemic.

The aim of this study is to analyze differences in participation, and in the results obtained in the anatomy and histology exams, over two academic years of the Sport Sciences degree course. During the first semester of the academic year 2019/2020 both the lectures and the exam took place face-to-face, while during the academic year 2020/2021 everything was done online. Statistical analysis revealed that the online modality was especially advantageous for the anatomy exam. Students' opinions were also assessed through a short questionnaire. The results showed that teachers involved themselves in both groups. Students needed to interact socially with teachers and colleagues and to ask them questions. Even if the differences were not significant, the difference was greater for face-to-face students in most comparisons. Finally, the most common methods of peer communication were by social media.

Berberine affects mitochondrial activity and cell growth of leukemic cells from chronic lymphocytic leukemia patients.

B-cell chronic lymphocytic leukemia (CLL) results from accumulation of leukemic cells that are subject to iterative re-activation cycles and clonal expansion in lymphoid tissues. The effects of the well-tolerated alkaloid Berberine (BRB), used for treating metabolic disorders, were studied on ex-vivo leukemic cells activated in vitro by microenvironment stimuli. BRB decreased expression of survival/proliferation-associated molecules (e.g. Mcl-1/Bcl-xL) and inhibited stimulation-induced cell cycle entry, irrespective of TP53 alterations or chromosomal abnormalities. CLL cells rely on oxidative phosphorylation for their bioenergetics, particularly during the activation process. In this context, BRB triggered mitochondrial dysfunction and aberrant cellular energetic metabolism. Decreased ATP production and NADH recycling, associated with mitochondrial uncoupling, were not compensated by increased lactic fermentation. Antioxidant defenses were affected and could not correct the altered intracellular redox homeostasis. The data thus indicated that the cytotoxic/cytostatic action of BRB at 10-30 µM might be mediated, at least in part, by BRB-induced impairment of oxidative phosphorylation and the associated increment of oxidative damage, with consequent inhibition of cell activation and eventual cell death. Bioenergetics and cell survival were instead unaffected in normal B lymphocytes at the same BRB concentrations. Interestingly, BRB lowered the apoptotic threshold of ABT-199/Venetoclax, a promising BH3-mimetic whose cytotoxic activity is counteracted by high Mcl-1/Bcl-xL expression and increased mitochondrial oxidative phosphorylation. Our results indicate that, while CLL cells are in the process of building their survival and cycling armamentarium, the presence of BRB affects this process.

Systemic inflammation and neurodegeneration in a mouse model of multiple sulfatase deficiency.

Sulfatases are involved in several biological functions such as degradation of macromolecules in the lysosomes. In patients with multiple sulfatase deficiency, mutations in the SUMF1 gene cause a reduction of sulfatase activities because of a posttranslational modification defect. We have generated a mouse line carrying a null mutation in the Sumf1 gene. Sulfatase activities are completely absent in Sumf1(-/-) mice, indicating that Sumf1 is indispensable for sulfatase activation and that mammals, differently from bacteria, have a single sulfatase modification system. Similarly to multiple sulfatase deficiency patients, Sumf1(-/-) mice display frequent early mortality, congenital growth retardation, skeletal abnormalities, and neurological defects. All examined tissues showed progressive cell vacuolization and significant lysosomal storage of glycosaminoglycans. Sumf1(-/-) mice showed a generalized inflammatory process characterized by a massive presence of highly vacuolated macrophages, which are the main site of lysosomal storage. Activated microglia were detected in the cerebellum and brain cortex associated with remarkable astroglyosis and neuronal cell loss. Between 4 and 6 months of age, we detected a strong increase in the expression levels of inflammatory cytokines and of apoptotic markers in both the CNS and liver, demonstrating that inflammation and apoptosis occur at the late stage of disease and suggesting that they play an important role in both the systemic and CNS phenotypes observed in lysosomal disorders. This mouse model, in which the function of an entire protein family has been silenced, offers a unique opportunity to study sulfatase function and the mechanisms underlying lysosomal storage diseases.

CD85j (leukocyte Ig-like receptor-1/Ig-like transcript 2) inhibits human osteoclast-associated receptor-mediated activation of human dendritic cells.

Immature dendritic cells (DCs) derived from freshly isolated human monocytes were used to evaluate the effect of the inhibiting receptor CD85j (leukocyte Ig-like receptor-1/ILT2) on activation induced by cross-linking of the human osteoclast-associated receptor (hOSCAR). CD85j and hOSCAR were expressed consistently at the same density on monocytes and on monocyte-derived DCs (both immature and mature). Cross-linking of hOSCAR, which activates via the FcR-associated gamma-chain, induced Ca(2+) flux in DCs. Concomitant cross-linking of anti-CD85j mAb abolished this early activation event. Likewise, CD85j stimulation strongly reduced IL-8 and IL-12 production by hOSCAR-activated DCs. Inhibition of DCs via CD85j also impaired their ability to enhance Ag-specific T cell proliferation induced by hOSCAR. Finally, because hOSCAR prevents apoptosis of DCs in the absence of growth/survival factors, CD85j cross-linking was able to counteract completely this antiapoptotic effect and to reduce Bcl-2 expression enhanced by hOSCAR stimulation. Thus, CD85j is an inhibiting receptor that is functional in human DCs.

Death of T cell precursors in the human thymus: a role for CD38.

Thymic T cell maturation depends on interactions between thymocytes and cells of epithelial and hematopoietic lineages that control a selective process whereby developing T cells with inappropriate or self-reactive receptors die. Molecules involved in this process are the TCR expressed on thymocytes together with the CD3 complex and MHC-peptide on accessory cells. However, other molecules may favor or prevent death of thymocytes, thus playing a role in selection. CD38 is expressed by the majority of human thymocytes, mainly at the double-positive (DP) stage. In contrast, CD38 is not found on subcapsular double-negative (DN) thymocytes and on a proportion of medullary single-positive (SP) thymocytes. CD38 enhances death of thymocytes when it is cross-linked by goat anti-mouse (GAM) antiserum or by one of its ligands, CD31, expressed by thymic epithelial cells or transfected into murine fibroblasts (L cells). As most thymocytes are at an intermediate (DP) stage of development, it is likely that these cells are most vulnerable to death mediated via MHC-peptide-TCR interactions that is increased by CD38 cross-linking. DN and SP thymocytes are refractory to CD38-induced apoptosis. Accessory molecules, e.g. CD38, are expressed during thymic cell maturation and their presence is relevant for the survival or death of DP T cells in the course of selection. Based on our data, CD38 enhances thymocyte death by interacting with CD31 expressed by accessory cells. In addition, CD28 expression on developing thymocytes also appears to play a role for their selection and it synergizes with CD38 to induce apoptosis of DP thymocytes.